ISGylation of DRP1 closely balances other post-translational modifications to mediate mitochondrial fission

Dynamin related protein 1 (DRP1), a pivotal mitochondrial fission protein, is post-translationally modified by multiple mechanisms. Here we identify a new post-translational modification of DRP1 by the ubiquitin-like protein, interferon-stimulated gene 15 (ISG15). DRP1 ISGylation is mediated by ISG15 E3 ligase, HERC5; this promotes mitochondrial fission. DeISGylation of DRP1 however leads to hyperfusion. Heterologous expression of SARS-CoV2 PLpro, a deISGylating enzyme, results in similar mitochondrial filamentation, significant decrease in total DRP1 protein levels and efflux of mtDNA. We report that deISGylated DRP1 gets ubiquitylated and degraded by TRIM25, instead of PARKIN and MITOL. While the cytosolic pool of DRP1 is primarily ISGylated, both mitochondrial and cytosolic fractions may be ubiquitylated. It is known that phosphorylation of DRP1 at S616 residue regulates its mitochondrial localisation; we show that ISGylation of phospho-DRP1 (S616) renders fission competence at mitochondria. This is significant because DRP1 ISGylation affects its functionality and mitochondrial dynamics in Alzheimer’s disease pathophysiology.


Cell culture and transfection
A549 (lung adenocarcinoma-derived hypotriploid alveolar basal epithelial cells) and HepG2 (human hepatoma cell line) cell lines were used.Maintenance of cells in culture was as before (76).Briefly, cells were grown in 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA)/Dulbecco's modified Eagle's medium (DMEM; Himedia, Mumbai, India) at 37°C and 5% CO2.A549, gift of Amit Pal (Kolkata, India); HepG2 cells, gift of Soumen Kanti Manna (Kolkata, India) were grown under standard cell culture conditions.For transfections of cells, Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) was used as per the manufacturer's instructions.24h post-transfection, cells were lysed in suitable buffers.All tissue culture plasticware used for microscopy were from Nunc, Rosklide, Denmark, and bottom coverglass dishes used were from SPL Lifesciences, Gyeonggi-do, Korea.

Brain lysates of human and mice
Human brain whole tissue lysates of normal (adult whole normal; NB820-59177) and AD (adult whole Alzheimer's; NB820-59363) were from Novus Biologicals.5XFAD transgenic mice brain lysates were gifted by S. C Biswas (Kolkata, India).
siRNAs were transfected using Lipofectamine 2000 following the manufacturer's instructions.For knockdown studies, cells were harvested 72 hours post transfection.Cells were transfected 48h post-treatment with siRNAs, if required.

Immunocytochemistry
For immunocytochemistry, cells were fixed with either 4% formaldehyde or methanol as per the requirement of the antibody, like before (76).Cells were permeabilized using 10% FBS/PBS/0.1% saponin (Sigma-Aldrich) for 60 min, followed by overnight staining in primary antibody at 4°C and 60 min incubation in secondary antibody at room temperature.
The samples were then imaged using confocal microscopes.

Western blotting and immunoprecipitation
The protocol for western blotting was as described before (77,78).10% or 12% Tris-tricine gels were used for SDS PAGE followed by Western blotting.Quantification of Western blots was done using Gel Quant and Image J softwares.At least 3 independent experiments were performed and band intensities were normalized to loading control.p-values were determined using Student's t-test.For immunoprecipitation, cells were lysed in immunoprecipitation buffer (50mM Tris-Hcl, pH 7.5, 150mM NaCl, 0.1% Triton X-100, 1% IGEPAL, 1mM PMSF, protease inhibitor cocktail (Sigma Aldrich), and immunoprecipitation was performed under denaturing conditions as described before.

Confocal imaging and image analyses
Confocal imaging was done using the Zeiss LSM980, Nikon A1R+ Ti-E microscope system.
Ar-ion laser (for GFP excitation or Alexa-Fluor 488 with the 488 nm line), a He-Ne laser (for RFP, Alexa-Fluor 594 excitation with the 561 line were used with 100×0.55NA oil immersion objective and 60×1.4NA water immersion objective.He-Ne laser for Alexa-Fluor 633 with the 633 line was also used with the same objective.Cells were imaged in CO 2 independent media maintaining conditions of live-cell imaging as described before (77).Cells were imaged taking z-stacks with z interval of 0.15 μm.Image analyses and 3D projections were done in FIJI.Quantitative analysis was done for 50-200 cells (as indicated) from more than 3 independent experiments.
Mitochondrial length measurement was done as discussed before (2).For TFAM puncta calculation, z-stack images were projected in 3D.TFAM puncta outside TOMM20 boundary was calculated manually.
Mitochondrial volume and number measurement were done using ImageJ (Mitochondria Analyzer).

3D structured illumination microscopy (3D-SIM)
3D-SIM images were obtained using Nikon N-SIM on Eclipse

Preparation of cytosolic and mitochondrial fractions
Cells were lysed in mitochondrial isolation buffer (10 mM HEPES (Sigma Aldrich, H3375)-KOH buffer [pH 7.4] containing 0.22 M mannitol (Sigma Aldrich, M4125), 0.07 M sucrose (Sigma Aldrich, S8501), by passing through a 25G needle attached to a 1ml syringe at least 10 times.A small fraction of this was saved as the whole-cell lysate.This was centrifuged at 600g to pellet unlysed cell debris and nuclear fractions.The remaining solution was centrifuged at 4000g to get the mitochondria enriched fraction, which was then washed twice with isolation buffer.The supernatant was collected as the cytosolic fraction.

Semi-permeabilization assay
Biochemical fractionation by selective detergent extraction has been described before (79).In brief, cells were washed with PBS twice and treated with PBS containing digitonin (at 100 ug/ml; 78) to generate the soluble cytosolic fraction.After collecting the cytosolic fraction, cells were scraped off the dishes with lysis buffer (50mM Tris pH 7.5, 150mM NaCl, 2mM EDTA, 0.5% TritonX-100, 0.5% Sodium deoxycholate) and centrifuged at 14200 RPM for 15 minutes.The supernatant was used as the membrane fraction.The cytosolic fractions were TCA precipitated, analyzed by SDS-PAGE, followed by immunoblotting against the indicated antibodies.
Calculations were done as before (81).

Statistical analyses
For all experiments, at least 3 biological replicates were used unless otherwise indicated.

ATP measurements
The protocol for ATP measurement adapted from SCENITH (Single Cell ENergetIc metabolism by profiling Translation inhibition) (82).For this, cells were plated at 1x 10 5 cells/ml, 0.2ml/well in 96-well plates for studying metabolism.Experimental duplicates/triplicates were performed in all conditions.After transfection or treatment, of cells, wells were treated during 45-60 minutes with DMSO, 2-Deoxy-D-Glucose (DG, final concentration 100mM), Oligomycin A (Oligo, final concentration 1μM), or a sequential combination of the drugs at the final concentrations.Cells without any cells were used as negative control.After treatment, ATP produced in cells was detected using Luminescent ATP detection assay kit as per manufacturer's protocol.Briefly, cells were lysed, substrate buffer added and luminescence was measured using BioTek SYNERGY HTX Reader at 540nm wavelength.

Supplementary figure legends
cytosolic fraction was used to purify mtDNA as per the manufacturer's instructions [DNA Blood and Tissue kit (Qiagen)].The pellet was used to isolate genomic DNA.After DNA isolation the samples were used for qPCR with mtDNA specific primers; Graphs have been plotted by exporting data of ImageJ to MS Excel 2007.Error bars represent mean±SEM, 2-tailed type 3 Students t-test has been used for calculating statistical significance of experiments.Boxplots have been generated using BoxplotR.

Figure S1 .
Figure S1.ISG15 mediated post-translational modification of DRP1 affecting mitochondria.(A) Reverse IP of samples corresponding to Figure1 panel A.  indicates band of interest (ISG15).(B) Reverse IP of samples corresponding to Figure1 panel B.  indicates band of interest (HERC5).(C) Graphs plot quantification of total mitochondrial volume (Z-stacks were taken with 0.15μm slices) and total mitochondria/cell.Data correspond to Figure1 panel E. ~150 cells from 3 independent experiments were analyzed.** p≤0.01, *** p≤0.001 using unpaired 2-tailed Student's t-test.(D) Cells transfected with either control vector or HA-tagged ISG15 were treated with MitoTracker Green FM and imaged under live-cell conditions.Scale bar, 20μm.Overexpression was confirmed by immunoblotting against ISG15, β-TUBULIN was used as loading control.(E) Box plot showing quantification of mitochondrial length for the experiment described in panel D. ~75 cells from 3 independent experiments were analyzed.The central line and the plus (+) symbol in each box show the median and mean value, respectively.*** p≤0.001 using unpaired 2tailed Student's t-test.(F) Cells treated with hIFNα1 similar to as described in Figure1 panel F were lysed and analysed for ISGylation of DRP1 by co-immunoprecipitation between ISG15 and DRP1.Note that presence of hIFNα1 induces enhanced ISGylation of DRP1.Further, partial rescue of endogenous DRP1 ISGylation was observed in mutant samples upon hIFNα1 treatment.ISG15 and VINCULIN served as loading controls. ISGylated endogenous DRP1,  ISG15-modified mCherry-tagged DRP1,  endogenous DRP1,  mCherry-tagged DRP1.(G) Graphs plot quantification of total mitochondrial volume (Zstacks were taken with 0.15μm slices) and total mitochondria/cell.Data correspond to Figure1 panel G. ~200 cells from 3 independent experiments were analyzed.** p≤0.01, *** p≤0.001 using unpaired 2-tailed Student's t-test.(H) Cells treated with mock or ISG15 siRNAs were imaged under live-cell conditions with MitoTracker Green FM.Scale bar, 20μm.Knockdown efficiency was confirmed by immunoblotting against ISG15, β-TUBULIN was used as loading control.Box plot showing quantification of mitochondrial length for the experiment.~75 cells from 3 independent experiments were analysed.The central line and the plus (+) symbol in each box show the median and mean value, respectively.*** p≤0.001 using unpaired 2-tailed Student's t-test.(I) Graphs plot quantification of total mitochondrial volume (Z-stacks were taken with 0.15μm slices) and total mitochondria/cell.Data correspond to panel H. ~70 cells from 3 independent experiments were analyzed.*** p≤0.001 using unpaired 2-tailed Student's t-test.

Figure S2 .
Figure S2.CoV-2 PLpro alters mitochondrial morphology and function.(A) Graphs plot quantification of total mitochondrial volume (Z-stacks were taken with 0.15μm slices) and total mitochondria/cell.Data correspond to Figure2 panel A. ~90 cells from 3 independent experiments were analyzed.ns, not significant (p> 0.07), *** p≤0.001 using unpaired 2-tailed Student's t-test.(B) Graph plots total ATP produced in A549 cells transfected with indicated constructs; a.u., arbitrary units.Data from 3 independent experiments were analyzed.** p≤0.01 using unpaired 2-tailed Student's t-test.(C) Histogram plots percentage of ATP produced corresponding to glucose dependence, FAO (fatty acid oxidation) and AAO (amino acid oxidation) capacity, mitochondrial dependence and glycolytic capacity of cells transfected with indicated constructs.Data from 3 independent experiments were analyzed.** p≤0.01 using unpaired 2-tailed Student's t-test.(D) HepG2 cells transfected with the indicated GFP-tagged constructs were imaged under live-cell conditions with MitoTracker Red FM.Immunoblot of lysates post imaging were probed against GFP and β-ACTIN to confirm transfection efficiency and similar loading.Scale bar, 5μm.(E) Box plot showing quantification of mitochondrial length for the experiment described in panel D. ~50 cells from 3 independent experiments were analyzed.The central line and the plus (+) symbol in each box show the median and mean values, respectively.*** p≤0.001 using unpaired 2tailed Student's t-test.(F) Transfected cell lysates of the indicated GFP-tagged constructs were immunoprecipitated and immunoblotted.Note reduced ISGylation of MFN2 in CoV2 PLpro WT samples.The input levels of MFN2 and β-ACTIN in the total lysates served as loading controls.(G) Cells transfected and processed similarly as in Figure2 panel E were imaged in the slice 3D-SIM mode.White boxes show insets.White arrowheads mark TFAM puncta outside TOMM20 boundary.Immunoblot of lysates post imaging probed against GFP and β-ACTIN confirm transfection and similar loading.Scale bar, 10μm.(H) Cells transfected with indicated constructs were loaded with LIVE ORANGE mito and PicoGreen to visualise cristae and dsDNA, respectively, and imaged in the slice 3D-SIM live mode.Enlarged views of the areas within the white boxes shown (insets).White arrowheads mark PicoGreen puncta outside LIVE ORANGE mito boundary.Scale bar, 10μm.

Figure S3 .
Figure S3.Cell line independent mtDNA release in presence CoV2 PLpro and activation of IFN-1 response pathway.(A) HepG2 cells were transfected with indicated GFP-tagged constructs and immunostained with antibodies against TOMM20 and TFAM.Representative images were taken in Z-stacks (0.15μm slices), images show 3-D projections.Enlarged views

Figure S4 .
Figure S4.HERC5 knockdown affects mitochondrial dynamics and mtDNA release.(A) Cells treated with mock or HERC5 siRNA pools were imaged under live-cell conditions with MitoTracker Green FM.Scale bar, 20μm.Knockdown efficiency was confirmed by immunoblotting against HERC5, VINCULIN was used as loading control.(B) Box plot

Figure S5 .
Figure S5.Post-translational modifications of DRP1, PARKIN and MITOL.(A) A549 cell lysates generated similarly as in Figure 4 panel B were verified for DRP1 ubiquitylation by endogenous Ub. Lysates were co-immunoprecipitated and probed against DRP1 and Ub.Note, increased DRP1 ubiquitylation in CoV2 PLpro WT samples.DRP1 and VINCULIN levels in the total lysates served as loading controls. marks the prominent ubiquitylated form of DRP1.(B) Cells co-transfected with CoV2 PLpro WT along with DRP1 or its mutant were co-immnunoprecipitated similar to Figure 4 panel E. Note decrease in ubiquitylated DRP1 detected in presence of the K532R mutant when compared with the mCherry-tagged wild type construct.VINCULIN served as loading control. endogenous DRP1,  mCherry-tagged DRP1.(C) A549 cells were transfected with indicated GFP-tagged constructs were immunoblotted with anti-DRP1 antibody.Note unchanged DRP1 levels upon PARKIN overexpression.Input levels of GFP and β-TUBULIN served as loading controls. Cytosolic GFP,  CoV2 PLpro GFP,  PARKIN GFP.(D) Cells were similarly transfected as panel C, except MITOL GFP was used instead of PARKIN GFP.Input levels of GFP and β-TUBULIN served as loading controls. Cytosolic GFP,  CoV2 PLpro GFP,  MITOL GFP.(E) A549 cells were transfected with indicated constructs along with

Figure S6 .
Figure S6.TRIM25 regulates DRP1 stability and mitochondrial dynamics.(A) A549 cells were transfected with control vector or TRIM25 GFP, and were either left untreated or and treated with cycloheximide (Chx, 100 μg/ml) for indicated time periods.DRP1 protein levels in cell lysates was analysed.β-ACTIN was used as loading control. endogenous TRIM25,  GFP-tagged TRIM25.Graph shows quantification of DRP1 levels over time.Data represents 3 independent experiments.ns, not significant (p=0.12),*p≤0.033,**p≤0.002,***p≤0.001using 2way ANOVA with Bonferroni corrections.Error bars, ±SEM.(B) Reverse IP of samples corresponding to Figure 4 panel H.  marks the band corresponding to DRP1.(C) Cell treated with mock or TRIM25 siRNA pools were coimmunoprecipitated against Ub and DRP1.Note reduced ubiquitylated DRP1 levels in TRIM25 knockdown samples and increased DRP1 levels in cell lysates of the same.TRIM25 levels in the lysates confirm knockdown efficiency; VINCULIN served as loading control.(D) Cells treated with mock or TRIM25 siRNA pools were imaged under live-cell conditions with MitoTracker Green FM.Scale bar, 20μm.Knockdown efficiency was confirmed by immunoblotting against TRIM25; VINCULIN was used as loading control.(E) Box plot showing quantification of mitochondrial length for the experiment in panel C. ~40 cells from

Figure S8 .
Figure S8.Altered mitochondrial function in Alzheimer's disease.(A) Graph plots total ATP produced in SHSY5Y cells transfected with indicated constructs and treated with DMSO or 0.5μM Aβ for 24h; a.u., arbitrary units.Data from 3 independent experiments were analyzed.** p≤0.01 using unpaired 2-tailed Student's t-test.(B) Histogram plots percentage of ATP produced corresponding to glucose dependence, FAO (fatty acid oxidation) and AAO (amino acid oxidation) capacity, mitochondrial dependence and glycolytic capacity of cells analysed in panel A. Data from 3 independent experiments were analyzed.* p≤0.05, *** p≤0.001 using unpaired 2-tailed Student's t-test.(C) Graph plots total ATP produced in SHSY5Y cells treated with DMSO or 1μM Aβ for 24h; a.u., arbitrary units.Data from 3 independent experiments were analyzed.*** p≤0.001 using unpaired 2-tailed Student's t-test.(D) Histogram similar to panel B plotted with data corresponding to panel C. ** p≤0.01, *** p≤0.001 using unpaired 2-tailed Student's t-test.

Quantitative reverse transcription and real-time PCR
Ti Inverted Microscope equipped with a Piezo stage, a Plan Apochromat 100x (NA 1.49) oil-immersion objective and